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Wikl-lype

CytOSOlic-sacB

Wikl-lype

CytOSOlic-sacB

Figure X. SKMs of March granules isolated from wild-type and transgenic seeds containing the cylosolic-targeled SacB gene. Wild-type and transgenic seeds were harvested from the same heterozygous plain.

expression of invertase genes, many plants also contain genes coding for a protein that specifically binds to and inhibits invertase activity [71-73], Glucose levels in mature seeds containing the cytosolic-targeted SacB gene were only slightly increased compared to wildtype (Wild-type = 2.27 mg g"1; SacB = 3.07 mg g"1, based on kernel dry weight). However, one mole of glucose is produced for every mole of fructose polymerized and the molar equivalence of glucose did not match expected levels in transgenic seeds. Fructan levels ranged from 16 to 18 mg g"', but glucose in mature seeds was 5 to 6-fold lower than expected. Thus, a large proportion of glucose released by the enzyme during fructose polymerization was metabolized and did not contribute to dry matter accumulation. It is not, however, clear whether glucose per se may be responsible for altered gene expression, leading to the severe changes in maize endosperm development.

Nevertheless, sugar regulation of gene expression must be coordinated with the growth and developmental needs of plants and is likely to be influenced by multiple signaling pathways. Signaling pathways may be influenced by changes in sucrose or hexose levels. Increased sucrose levels have been shown to regulate gene expression in plants [74-76], The effect of glucose on gene expression has also been established and is better known from work with several fungal species [77], The first enzymatic step in metabolism of glucose is addition of phosphate mediated by hexokinase. Hexokinase appears to play a role in sensing sugar levels in cells, and may be involved in signal transduction leading to changes in gene expression [77].

Hexokinase has recently been implicated as a sugar sensor, mediating gene expression in plants, but this dual role has not yet been firmly established [68]. An alternate sensing mechanism involving the ratio of AMP to ATP has been suggested for recognition of cell energy status leading to changes in gene expression [78], It is, therefore, interesting to note that a substantial portion of hexokinase activity in maize is associated with mitochondria [79].

The physical attachment of hexokinase to the outer membrane of mitochondria has been extensively studied in mammalian cells where hexokinase type I was shown to form a complex with a porin and the adenylate translocator [80]. This complex couples hexokinase to internal ATP and may function as a sensor for the metabolic state of the cell [81]. Sensing the metabolic state of mammalian cells is mediated by numerous effectors, including ATP. Changes in the ATP/ADP ratio can cause collapse of the mitochondrial membrane potential that in turn induces programmed cell death [82]. In light of this connection, it is not unreasonable to consider that expressing the SacB gene in transgenic maize leads to an altered metabolic state due to increased glucose production. The altered metabolic status of the cell, recognized by hexokinase bound to mitochondria, may then lead to premature PCD and could explain the phenotype demonstrated in Figure 7.

Rober et al. [83] also found that compartmentation of an Erwinia amylovora fructosyltransferase was influential to development in transgenic potato tubers. Tuber-specific expression of the fructosyltransferase resulted in fructan accumulation and increased glucose levels. They also found that starch synthesis was reduced in the transgenic tubers and suggested that glucose accumulation may be related to this phenotype. One possible explanation given was that high levels of glucose in tubers leads to repression of endogenous gene expression, particularly sucrose synthase, which is the first enzymatic step in the starch biosynthetic pathway. Similar results were reported when a yeast-derived invertase gene was expressed in transgenic potato tubers [84], Cytosolic expression of the yeast gene led to a decrease in both tuber dry weight and starch yield. Tuber specific expression of invertase in the cytosol also led to increased glucose production and a three-fold increase in enzymes involved in the respiratory pathway [85]. Fructose is also a product of the invertase reaction, but does not accumulate in transgenic tubers. Redirection of sucrose away from starch synthesis was attributed to glucose induced signaling of genes in the respiratory pathway [86]. Free glucose has previously been shown to induce changes in gene expression, leading to altered plant development [70], Studies using 14C-labelled glucose and fructose in potato tuber disks also demonstrate a significant difference in the metabolism of the two different hexose sugars [87], Equilibrium of the sucrose synthase reaction was shifted by the addition of fructose resulting in sucrose cycling. Glucose did not alter partitioning into sucrose, but greatly affected the rate of starch synthesis. Each of these examples show that glucose and fructose affect metabolism differently in plant cells. Production of glucose from sucrose, rather than polymer accumulation in transgenic seeds, is much more likely to be recognized by the cell as a condition requiring changes in gene expression.

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